HU219607B - Process for homocysteine assay and analitical kit - Google Patents

Abstract

There is provided a method for assaying homocysteine in a sample, e.g. blood, plasma or urine, which comprises the steps of contacting said sample with a homocysteine converting enzyme other than SAH-hydrolase and at least one substrate for said enzyme other than homocysteine, and without chromatographic separation assessing a non-labelled analyte selected from a homocysteine co-substrate and the homocysteine conversion products of the enzymic conversion of homocysteine by said enzyme.

Description

The present invention relates to a method for testing homocysteine in clinical samples.

Homocysteine is an intermediate amino acid that is formed when methionine is metabolized to cysteine. Generally, homocysteine formed in the body is rapidly converted in one of two ways: (1) condensed with serine to form a cystation, or (2) converted to methionine; and its concentration (and the concentration of the oxidized form of homocysteine) in the living organism under normal conditions are substantially negligible.

However, the concentration of homocysteine in biological samples may be of clinical significance in a number of cases because homocysteine plays an important role in the complex of biochemical reactions that make up the metabolism of sulfhydryl amino acids and its accumulation may indicate a variety of disorders in these biochemical reactions including metabolism. For example, it is known that homocystinuria (an abnormal formation of homocysteine in urine) is a disorder of amino acid metabolism resulting from the lack of enzymes of methyl tetrahydrofolic acid methyltransferase that catalyze the methylation of cystation-β-synthetase or homocysteine to methionine.

The metabolism of sulfhydryl amino acids is closely related to folic acid and vitamin B4 (cobalamin), which act as substrates or cofactors in the various transformations that occur. Therefore, accumulation of homocysteine may be an indicator of malfunctioning of cobalamin or folate-dependent enzymes, or other disorders or diseases related to cobalamin or folate metabolism.

In addition, since the conversion of homocysteine to methionine is based on a reaction requiring S-methyl tetrahydrofolate as the methyl donor, homocysteine metabolism may be affected by other antifolate drugs, such as methotrexate, for the treatment of cancer. Therefore, the detection of homocysteine is also indicated in the treatment of cancerous diseases with antifolate agents.

Today, elevated levels of homocysteine in the blood are associated with the development of atherosclerosis (Clarke et al., New England J. Med. 324, 1149-1155 (1991)), and even mild homocysteinemia is considered a risk factor for the development of cardiovascular disease. Therefore, the determination of homocysteine levels in plasma or blood is also of importance for the diagnosis of vascular disease.

Although no immunological methods are available for the direct determination of homocysteine due to the lack of antibodies against homocysteine, many other methods are known for the determination of homocysteine in clinical samples. They all require chromatographic separation and are generally based on one of the following three principles:

(1) classic chromatographic amino acid analysis;

(2) reacting homocysteine in the sample with S-adenosyl-L-homocysteine hydrolase in the presence of a radiolabeled or otherwise labeled S-adenosine cosubstrate, and isolating and quantifying the product formed (S-adenosyl-L-homocysteine, SAH): generally, chromatographic separation (HPLC or TLC) and radioactivity measurement are used (see Refsum et al., Clin. Chem., 31, 624-628 (1985); Kredich et al., Anal. Biochem. 116: 503-510 (1981); Chui, Am. J. Clin. Path. 90 (4): 446-449 (1988); Totani et al., Biochem. Soc. 14 (6): 1172-1179 (1988); and Schimizu et al., Biotechnol. Appl. Biochem. 8: 153-159 (1986)];

(3) reacting the homocysteine in the sample with fluorophore followed by HPLC separation and fluorimetry [Refsum et al., Clin. Chem. 35 (9): 1921-1927 (1989).

These procedures are time-consuming and cumbersome, and are all based on direct quantification. More specifically, a known feature of the known processes is chromatographic separation, which requires highly specific and complicated equipment.

The use of such devices is generally undesirable in routine clinical laboratory practice and is generally not suitable for automation in routine clinical laboratory procedures.

Therefore, there is a need for an improved assay for homocysteine that is simple, specific, fast to implement, easy to adapt to clinical laboratory use, and, above all, does not require expensive and time-consuming chromatographic separation. The object of the present invention was to provide a process meeting the above requirements.

It is therefore an object of the present invention to provide a method for assaying homocysteine in a sample by contacting the sample with a homocysteine-converting enzyme such as Sadenosyl Homocysteine (SAH) hydrolase and at least one substrate other than homocysteine of the enzyme and chromatographing the reagents or reaction products. (preferably photometrically) determining an unlabeled substance to be determined which may be a reaction product of the homocysteine co-substrate or the enzymatic conversion of homocysteine by said enzyme.

Preferably, the sample is incubated for at least 30 seconds, more preferably at least 5 minutes after mixing with the homocysteine converting enzyme and the substrate before proceeding to the next step of the assay.

SAH hydrolase is preferably used as the homocysteine converting enzyme in the process of the invention, but other enzymes may also be used. Examples include betaine homocysteine methyltransferase and other enzymes involved in the conversion of homocysteine. See, e.g., Graham, Trends Cardiovasc. Med. 1: 244-249 (1991).

The homocysteine co-substrate as defined in the process of the present invention is a compound that reacts with homocysteine in an enzyme-catalyzed homocysteine conversion reaction, such as SAH hydrolase.

Definition as used herein includes both quantitative and qualitative definitions, that is, either an ab2

An absolute value is obtained for the amount or concentration of the analyte present in the sample, such as homocysteine co-substrate, or an index, ratio, percentage, visual or other value indicating the concentration of the analyte in the sample. The assay may be direct or indirect, and the chemical compound actually detected is, of course, not necessarily the substance to be determined, but a derivative thereof or other substance as described below.

The assay of the present invention preferably utilizes either an enzymatic or immunological method to determine the substance to be determined. According to a preferred enzymatic process, the substance to be determined is contacted with another enzyme having the substrate to be determined and either a co-substrate or a direct or indirect reaction product of the enzymatic conversion of the substance to be determined by this enzyme. it consists of the competitive binding of an additional hapten (e.g., a polyhapten or a labeled analog of the substance to be assayed) to the antibody, and determining the bound or unbound hapten.

The preferred homocysteine converting enzyme used in the present invention is S-adenosyl homocysteine hydrolase (SAH hydrolase), which catalyzes the following reaction of homocysteine:

adenosine + homocysteine S-adenosyl homocysteine (SAH), reaction equilibrium constant (K.) 10 «»-.

The reaction can take place in both directions, depending on the reaction conditions, the concentration of reactants, and so on. Depending on.

In the above reaction, adenosine is a co-substrate of homocysteine. Other co-substrates such as adenosine analogs or related compounds may also be used in the present invention.

A major advantage of the assay of the invention is that homocysteine acts as an inhibitor of SAH hydrolase, inhibits the hydrolytic reaction leading to the production of homocysteine and adenosine, and shifts the equilibrium reaction towards SAH synthesis.

The amount of homocysteine present in the sample thus has a direct effect on the production or consumption of homocysteine substrate, such as adenosine, by SAH hydrolase, and thus its concentration in the reaction mixture. According to the invention, the concentration or change in the concentration of a homocysteine co-substrate, such as adenosine, in the reaction mixture can be used as an indicator of the initial concentration of homocysteine in the sample. Therefore, when the substance to be determined is the co-substrate, the assay of the present invention differs from known methods in that, instead of directly determining homocysteine, homocysteine is indirectly determined by determining the concentration of its co-substrate during enzyme-catalyzed conversion. The direct advantage of this is that the methods used in conventional clinical laboratory procedures, such as photometric methods, which are not applicable in known homocysteine assays, are applicable, making the method of the invention particularly suitable for routine clinical trials.

In a preferred embodiment of the assay of the invention, the SAH hydrolase reaction can be used in both directions. Thus, by contacting the test sample with adenosine and SAH hydrolase, a portion of the adenosine corresponding to the amount of homocysteine consumed is consumed and the amount of homocysteine in the sample can thus be determined by varying the concentration of adenosine. Alternatively, adenosine analogs and / or adenosine producing compounds may be used in place of adenosine.

In another preferred embodiment of the invention, the reverse reaction may be used. The test sample is contacted with SAH (usually in excess) and SAH hydrolase. Hydrolysis of SAH then produces homocysteine and adenosine. The homocysteine present in the test sample counteracts this reaction and thus inhibits the formation of adenosine, the amount of which is monitored.

SAH hydrolase substrates used in the process of the invention may be, for example, SAH or adenosine or analogs or precursors thereof.

Many enzymes are involved in complex pathways of sulfhydryl amino acid metabolism and transmethylation reactions in the body. These pathways and reactions have been thoroughly studied and the regulatory role of the enzymes involved has been investigated. The role of one such enzyme, SAH hydrolase, is summarized in Ueland (Pharmaceutical Reviews 34, 223-253 (1982)). Trewyn et al., J. Med. Biochem. Biophys. Met. 4, 299-307 (1981)] describes an assay for the regulatory role of SAH hydrolase and a method for assaying the enzymatic activity of SAH hydrolase. Garras et al., Analytical Biochem. 199, 112-118 (1991), describe other homocysteine pathways and more specifically describe an assay for homocysteine methionine synthase-mediated conversion. As mentioned above, Graham (supra) discloses further enzyme-catalyzed homocysteine transformations. The co-substrates and the products of the transformations of the various reactions described above can be used as assay agents in the assay of the invention, particularly when assayed by immunological means.

The clinical specimen to be tested by the method of the invention may be derived from any biological fluid or tissue extract and may be pretreated prior to testing. However, plasma or urine samples are generally used.

Significant amounts of homocysteine present in plasma or urine may be disulfide bonded to circulating proteins (e.g., albumin) and may also be present in the form of other disulfide derivatives (usually homocysteine-cysteine conjugates). It may therefore be desirable to treat the sample with a reducing agent to cleave disulfide bonds to determine the amount of total homocysteine in the sample.

EN 219 607 Β s and free homocysteine. The disulfides can be readily and specifically reduced with thiols (e.g., dithiothreitol (DTT), dithioerythritol (DTE), 2-mercaptoethanol, cysteine thioglycolate, thioglycolic acid, glutathione and the like). Direct chemical reduction may also be carried out with borohydrides (e.g. sodium borohydride) or amalgams (e.g. sodium amalgam), but more specific reagents such as phosphors or phosphorothioates may also be used. The reduction of the disulfide is described in detail by Jocelyn (1987) Methods in Enzymology 143: 243-256, listing a wide variety of suitable reducing agents.

Adenosine or other co-substrates of homocysteine may be determined by known methods. In general, photometric (e.g., colorimetric, spectrophotometric or fluorometric) detection and immunological methods are preferred as they are particularly readily adapted to clinical laboratory applications. Methods based on enzymatic reaction or reaction with mono- or polyclonal antibodies are particularly preferred because they are simple and rapid and relatively inexpensive to implement. For example, to measure the substance to be determined, the reaction is followed by enzymes which convert it directly or indirectly into products which can be detected photometrically, for example spectrophotometrically. Suitable enzymes which, of course, cannot react with other substrates of the homocysteine converting enzyme, especially homocysteine, include, for example, adenosine deaminase (which converts adenosine and ATP into ADP and phosphorylated adenosine). These enzymes can also be combined with other enzymes that convert the resulting product into other detectable products.

For example, an immunoassay may be a method in which a substance to be assayed reacts with its specific antibodies, which can be self-assayed or further reacted to produce detectable products, for example by sandwich assay. However, in a particularly spectacular immunological procedure, a fluorophore-labeled analog of the substance to be assayed, preferably a co-substrate such as fluorescein-labeled adenosine, is used and this and the unlabeled assay are reacted with an antibody to the assay. When the product obtained is subjected to fluorescence polarization assay using polarized gel-shining radiation, the concentration of the unlabeled analyte can be deduced from the degree of depolarization of the fluorescent radiation. Adenosine antibodies are commercially available (e.g., Paessel and Lorel GmbH, Frankfurt, Germany; and Serotech Ltd., Oxford, United Kingdom) and fluorescence polarization immunoassays (FPIA) are well known (see, for example, U.S. Patent Nos. A4420568 and US-A-4593089). and other Abbott Laboratories publications on TDx technology).

Detection reactions in the assay method of the invention include, for example:

antibody (1) adenosine + - labeled adenosine> FPIA detection + fluorescein (2) Adenosine -> _NH3 + cinema Adenosine deaminase (3) adenosine>mozin-> hypoxanthine adenosine purine phosphorylase nukleodezamináz zid-

O ₂ H ₂ O ₂ O ₂ H ₂ O ₂ —- = ¾ xanthine —- = ¾ uric acid xanthine xanthine oxidase oxidase (4) adenosine + ATP ->adenosine-5'-P + ADP adenosine kinase together with a competitive chemiluminescent ATP reaction such as luciferase

ATP + luciferin + O ₂ -> oxyluciferin + PPj + + AMP + CO ₂ + light or a fluorophore-labeled adenosine

It competes with ATP / adenosine kinase as determined by fluorescence polarization measurement.

As for Reactions (2) and (3), inosine and uric acid have characteristic UV absorption properties and can thus be determined spectrophotometrically by kinetic measurements.

However, there are some limitations to the UV detection of uric acid or inosine, since the sensitivity of the method is quite low and a UV light source and UV light transmitting sample holder are required. Therefore, it may be more appropriate to use colorimetric detection or electronic sensors, which are commonly used in clinical laboratories, particularly colorimetry.

In this regard, reaction (2) is particularly suitable since ammonia formed in the reaction catalysed by adenosine deaminase can be detected by known colorimetric methods. For example, ammonia formed in the sample can form colored products with appropriate reactants, the formation of which can be detected spectrophotometrically. One such method is that the ammonia is reacted with phenol in the presence of hypochlorite to give a colored indophenol derivative under basic conditions according to Scheme 1 (Bergmeyer, Methods of Enzymatic Analysis 1, 1049-1056 (1970)). The catalyst may be sodium nitrobutyrate. Modified versions of the process can also be used, for example, using various derivatives of phenol.

The amount of colored final product formed is directly proportional to the concentration of ammonia in the sample and therefore to adenosine.

In reaction (3), the xanthine oxidase reaction itself leads to detection using fluorogens or chromogens, such as redox indicators, by measuring the reduction / oxidation potential or by measuring the O ₂ consumption4.

EN 219 607 Β or more precisely by measuring the formation of H ₂ O ₂ , for example by using electronic sensors. A number of redox indicators can be used for this purpose and several methods for the determination of H ₂ O ₂ and O ₂ in solution are described in the literature. Indeed, H ₂ O _{2 is} frequently detected in clinical trials.

Suitable redox indicators include, but are not limited to, methylene blue, 2,6-dichlorophenol, indophenol, and the redox indicators listed in Table 1 of Kodak Laboratory and Research Products (Cat. No. 53). Redox reactions can be enhanced by the addition of enzymes with peroxidase activity, such as horseradish peroxidase.

When precipitating chromogen is required, MTT tetrazolium may be used in combination with xanthine oxidase or other similar enzyme. By using precipitating chromogen or fluorogen, immobilized color or fluorescence can be read as a visual indication of homocysteine concentration.

Reaction (4) uses a chemiluminescent ATP reaction to determine the concentration of adenosine. The great advantage of measurements based on chemiluminescence is that very low measurement thresholds are achieved and the equipment required is relatively simple. Chemiluminescence reactions can be used to detect various substances to be determined, such as ATP or H ₂ O ₂ , and one of the most effective and best known reaction is the flies bioluminescence reaction:

luciferase

ATP + luciferin + O ₂ -> oxyluciferin + PPj + + AMP + CO ₂ + light

Fire fly luciferin has a benzothiazole structure, but there are other biological sources of luciferins with different structures. For analytical purposes, ATP, luciferin and luciferase can be assayed directly using the above reaction. Another chemiluminescence reaction that can be used to form H ₂ O ₂ , for example in reaction (3), is the reaction of luminol (also known as 5-amino-2,3-dihydrophthalazine-1,4-dione) with hydrogen peroxidase catalyst , which produces light emission at 425 nm.

Hydrogen peroxide can also be determined, for example from reaction (3), by non-enzymatic chemiluminescence reactions of peroxy oxalate and acridinium esters (the latter in aqueous solution at neutral pH η).

The use of fluorophore-labeled adenosine in Reaction (4) and determination by fluorescence polarization measurement can be accomplished due to the relatively broad substrate specificity of adenosine kinase. This broad specificity is also suitable for compensating for endogenous adenosine (or other adenosine kinase nucleoside substrate) by adding adenosine kinase as a pretreatment, preferably in combination with a reducing agent (e.g. DTT).

The above enzymatic pretreatment of the sample is desirable since, in many of the embodiments of the invention, the amount of substance to be determined (e.g. adenosine) is already present in the sample in varying amounts, thus providing a potential source of error for the method. The background concentration of the substance to be determined can be eliminated by performing a portion of the sample without the use of a homocysteine-converting enzyme (e.g., SAH hydrolase); however, such a procedure is time-consuming and complicates the investigation. Alternatively, the sample is pretreated with an agent that transforms or removes the substance to be determined, for example, background adenosine is removed by an enzyme such as adenosine kinase or adenosine deaminase. As mentioned above, to avoid unnecessarily increasing the duration of the assay, such treatment of the sample may conveniently be accomplished by treating the sample with a reducing agent to release homocysteine.

In addition to the spectrometric or colorimetric methods used to measure the substance to be determined, other photometric methods may be used. Particularly suitable methods include particle agglutination and immunoprecipitation methods. When polyclonal antibodies are used, direct particle agglutination and immunoprecipitation may be used, although this is generally not advantageous when the homocysteine is released by SAH hydrolase. However, methods based on precipitation inhibition and particle agglutination inhibition may be used. They are based on antibody / hapten combinations which lead to precipitation or particle agglutination detectable by conjugation by turbidimetric or nephelometric measurements. In the case where the formation of the antibody / hapten complex is inhibited by a substance to be determined, such as SAH, the SAH content can be determined from the reduction in precipitation / aggregation. The reaction can be expressed as follows:

antibody + hapten -> complexation (optionally (preferably (precipitation or polyhaptene to particle) particle-bound) aggregation)

When the assay of the present invention is carried out using SAH hydrolase as the homocysteine converting enzyme, the SAH hydrolase is preferably stored in the presence of a reducing agent or treated with a reducing agent before use in the assay of the invention. It has also been found that storage leads to loss of SAH hydrolase activity and that the above application of the reducing agent either prevents storage inactivation or triggers reactivation of the enzyme prior to use.

Various reducing agents (e.g., DTT, cysteine, mercaptoethanol, dithioerythritol, sodium borohydride, etc.) may be used, but DTT, for example at a concentration of about 5 mM, is particularly preferred. The DTT itself should be stored at a low pH η, therefore the assay kit preferably contains a DTT solution at a low pH η (eg around pH 3) but with a low buffer capacity and separately contains the SAH hydrolase solution - which is partially or completely inactive may be - substantially neutral pH η and preferably buffered. When these solutions are combined, the enzyme is reactivated to a neutral pH η. The combination may, if desired, be carried out in the presence of a test sample or assay5

The sample is added shortly thereafter to simultaneously release the homocysteine. The other reducing agents mentioned above can likewise be used to stabilize / activate SAH hydrolase as well as to reduce the sample to release homocysteine.

The use of reducing agents to reactivate inactivated SAH hydrolase is also within the scope of the present invention. The invention also provides a kit comprising an inactive SAH hydrolase in one compartment and an acidic medium (e.g., at pH 3) containing a reducing agent such as DTT in another compartment. When using the kit, the SAH hydrolase can be mixed with the reducing agent and thus reactivated immediately before use in the assay.

Other additives may also be advantageously used to enhance the stability of the SAH hydroxylase during storage or in the assay itself. Examples of such additives are NAD +, glutathione, polyol alcohols and sugars (e.g. inositol, sorbitol, xylitol, erythritol, glycerol, ethylene glycol, sucrose, lactite, etc.), soluble polymers such as certain dextrans, and proteins (e.g. carrier proteins).

If antibodies are used in the assay of the invention, they may be polyclonal or preferably monoclonal. Where the desired antibodies are not commercially available, they can be prepared by known methods. Antibodies, monoclonal or polyclonal, can be raised in animals or hybridomas as described, for example, in James Gooding, Monoclonal Antibodies: Principle and Practice, Academic Press, London, 1983, Chapter 3. Monoclones must be classified in order to select those clones which are capable of distinguishing the desired hapten and other substrates of the enzyme (s), for example, that distinguish adenosine from SAH. Polyclonal antibodies that react only with the substance to be assayed (e.g., SAH) should be purified from cross-reacting antibodies, i.e., those that react with substrates other than the substance to be assayed (e.g., both adenosine and SAH). Purification can be accomplished by affinity chromatography, for example using immobilized adenosine when the substance to be determined is SAH.

For the production of antibodies, either the substance to be determined or another molecule containing the portion of the substance to be considered as the most appropriate binding region, such as a region far from the regions involved in the enzyme reaction, is used as the hapten. The hapten is preferably attached to a macromolecule such as BSA or hemicyanin. For SAH, the desired epitope is preferably the thioether, or near, it can be used itself as the (A) SAH formula conjugated to a macromolecule or, preferably, a formula "simplified" compound (I) - wherein R] and _R2 the same or different and hydrogen; or

-OK, (wherein R 4 is a lower aliphatic (e.g., C 1-6, especially C 1-4) aliphatic, e.g. alkyl, preferably methyl or ethyl), or

R 1 and R ₂ together represent oxygen, and

R ₃ is an amine or carboxyl residue or a salt or ester thereof, such as an ester with a C 1-4 alkanol, also attached to a macromolecule.

Examples of compounds of formula (I) which are useful as haptens include (I-1), (1-2), (1-3), (1-4), (1-5) and (1-6).

Such "simplified" constructs can also be applied to labeled analogs used in the above assays where a competitive binding reaction to the antibody occurs between the substance to be determined and the labeled analog. Thus, the signaling R * residue, which may be a fluorophore, chromophore, radioactive label, enzymatic, chemiluminescence, or other label commonly used in immunoassays, may be linked to the substance to be determined (such as R * -SAH) or the simplified epitope conjugated molecule (as in the compounds of formula (Γ)).

Of course, the above-labeled residues may also be linked to particles, polymers, proteins or other materials if desired.

The labeled furanose-6-thioethers and the compounds of the formula I are new and are also part of the invention.

In this respect, the present invention also relates to a process for the preparation of a compound of formula (I) wherein: (a) a compound of formula (II) wherein

R 1 and R ₂ are as defined above, and each R _{5 is a} protected hydroxy group, or two R _{5 taken} together form an alkylenedioxy group (i.e., a protected bis-hydroxy group such as -OC (CH ₃ ) ₂ ) O -] - With propyl halide of formula (III) - in the formula

R ₆ is R ₃ or a protected R ₃ and

Hal is a halogen atom such as a bromine atom, optionally deprotected, or (b) a compound of formula I wherein R ₃ is amino, a compound of formula II with acrylonitrile reacting and reducing the resulting cyanopropylthioether product and deprotecting and, if desired, (c) esterifying a compound of formula (I) wherein R ₃ is carboxyl. The starting compounds of formula III are known in the art or can be prepared by known methods. The starting compounds of formula (II) can be prepared from the corresponding 1-hydroxymethyl-furanoses by protecting the cis-hydroxyl group, bromination followed by reaction with thiourea, and by hydrolysis. The protection of the cis-hydroxyl group in 1- (hydroxymethyl) furanoses is

This can be done by reacting with reactants such as acetone.

Examples of compounds of formula (I) are:

The compounds of formulas (1) and (3) are commercially available.

UV absorption, visible light absorption, or fluorescence of substances in the reaction mixture, optionally precipitated or otherwise separated from the reaction mixture, can be determined at the end of the reaction (when the signal is stable) or at one or more specific times, or kinetic measurements more measurements.

The reactants required to carry out the assay of the invention are added sequentially or simultaneously to the reaction mixture. However, in many preferred embodiments, one or more reactions are preferably allowed to proceed for some time before the addition of the reactant required for the subsequent reaction (s). For example, when the test sample is reacted with adenosine and SAH hydrolase, it is preferable to allow the formation of SAH from homocysteine and adenosine to proceed for a while before proceeding with the determination of adenosine.

It is standard practice in clinical chemical analytical procedures to record standard curves for calibration purposes. Thus, in carrying out the method of the invention, known homocysteine-containing samples can be used to determine the response / measurable signal in place of the clinical samples to generate a standard curve, and then determine the homocysteine content of the unknown sample from the standard curve. Therefore, it is not necessary to determine the exact amount of the signaling molecule or redox potentials.

The assay of the present invention can be used to detect pathological or potentially pathological conditions associated with or manifesting in the homocysteine content of body fluids or tissues. This includes, for example, atherosclerosis, blood disorders, vitamin deficiencies and / or congenital malformations of metabolism. The assay of the invention can also be used to evaluate the effects of drugs such as antifolate agents.

The present invention also relates to an analytical product, optionally in kit form, for testing a homocysteine in a sample which

- a homocysteine-converting enzyme,

- from a substrate other than homocysteine of the enzyme,

a signaling agent, and

- where appropriate, a means of measuring the signal.

In one preferred embodiment, the analytical product is used to determine the homocysteine content of the sample

- a homocysteine converting enzyme such as Sadenosyl Homocysteine Hydrolase,

- one or more substrates other than homocysteine of the enzyme,

- means for producing a detectable derivative of a substance to be determined selected from the homocysteine co-substrate and the products of the enzymatic conversion of homocysteine, and

- optionally, means for spectrometric or colorimetric measurement of the detectable derivative.

In another embodiment, the article is

- adenosine,

- an adenosine converting enzyme,

optionally from a co - substrate of the adenosine converting enzyme, and

- comprising the aforementioned cosubstrate or products of the enzymatic conversion of adenosine by the above-adenosine converting enzyme, and means for generating a photometrically detectable response.

The adenosine converting enzyme is, for example, adenosine kinase and the means capable of producing a detectable response is, for example, ATP, luciferin and a luciferase. Alternatively, the adenosine converting enzyme may be adenosine deaminase and the means for conversion may be, for example, nucleoside phosphorylase, xanthine oxidase and a peroxidase.

In a further embodiment, the kit

- adenosine,

- S-adenosyl homocysteine,

- optionally, anti-S-adenosyl homocysteine antibody bound to matrix particles,

a polyhaptene suitable for the above antibody, and

- optionally, means for photometric determination of the agglutination or precipitation of antibody / polyhaptene complexes.

According to this embodiment, the polyhaptene may conveniently be a backbone polymer to which several furanose-6thioethers are attached, for example in the form of the side chains of formula (a).

For example, the above compounds are prepared by reacting a carboxylic acid of formula (I) or an anhydride or an acid halide thereof with a polymer having multiple doping amino groups (e.g., polylysine), or reacting an amine of formula (I) with a doping carboxylic acid polymer.

All or some of the reagents in the kit may be in the dry state as a form / matrix for performing reactions in the reaction mixture. Similarly, as mentioned above, the kit may include a relatively inexpensive spectrometric or colorimetric device for determining the detectable analyte or derivative, such as a light source and detector device for measuring luminous intensity at a typical wavelength of the detectable substance, etc., and even a simple colorimetric calibration card.

The invention is further illustrated by the following non-limiting examples. The test of Example 19 is particularly preferred.

Example 1

The sample (aqueous homocysteine solution for calibration or plasma or urine for clinical examination) is pretreated with a reducing agent (e.g., 10 mM dithiothreitol). This sample - preferably at a final concentration of 10 ^{6 to} 105 ⁵ mol / L homocysteine from 5 mg / ml rabbit

EN 219,607 Β of IgG, 20 milligrams / ml adenosine deaminase, 20 milligrams / ml nucleoside phosphorylase, 20 milligrams / ml xanthine oxidase, 500 milligrams / ml horseradish peroxidase, 10 mM dithiothreitol, 100 mM Add to a solution of sodium phosphate adjusted to pH 7.4 and 100 milliliters of S-adenosyl-1-homocysteine hydrolase at 37 ° C. Simultaneously, or sequentially, preferably after incubation for 10 minutes, adenosine is added to the sample at a final concentration of 5 x 10 ⁶ M to ensure conversion of adenosine in the sample. UV absorption is measured for 10 minutes and the response is measured kinetically at 292 nm and the AA / At value is calculated.

For clinical studies, the concentration of homocysteine can be calculated from a standard curve using known standards by interpolation.

Example 2

The sample (see Example 1) is pretreated with a reducing agent (e.g., 10 mM dithiothreitol). This sample, preferably at a final concentration of 10 ^{6 to} 10 ^{3 * 5} M homocysteine, contains 5 mg / ml of rabbit IgG, 20 milliliters / ml of adenosine deaminase, 20 milliliters / ml of nucleoside phosphorylase, 500 milliliters / ml of horseradish peroxidase , 10 mM dithiothreitol, 100 mM sodium phosphate adjusted to pH 7.4 and 20 milliliters of S-adenosyl-1-homocysteine hydrolase at 37 ° C. Simultaneously or sequentially, preferably after incubation for 10 minutes, adenosine is added to the sample at a final concentration of 5 x 10 ~ ⁵ mol / l to ensure conversion of adenosine in the sample. UV absorption is measured for 10 minutes and the response is measured kinetically at 292 nm and the AA / At value is calculated.

For clinical studies, the concentration of homocysteine can be calculated from a standard curve using known standards by interpolation.

Example 3

Assay Buffer: 0.1 M phosphate buffer (pH 7.4) containing 1 mg / ml rabbit IgG and 10 mM dithiothreitol.

Execution of the test

To 150 µl of assay buffer is added 3 µl of S-adenosyl-1-homocysteine hydrolase and a sample (preferably pre-treated according to Examples 1 and 2). The enzyme reaction is initiated by the addition of adenosine dissolved in assay buffer to a final concentration of 2.5 10 ⁵ . After incubation for 10 minutes, 20 µl of assay buffer containing 750 µl of adenosine deaminase, 20 µl of nucleoside phosphorylase, 20 µl of xanthine oxidase and 375 µl of horseradish peroxidase are added. UV absorption was measured kinetically at 292 nm for 5 minutes. Calculate the AA / At value. In parallel, the above assay was repeated but without the addition of S-adenosyl-1-homocysteine hydrolase. The difference between the two AA / At values is calculated and the concentration of homocysteine is determined from the standard curve by interpolation.

Example 4

The assay was performed as described in Example 3 until the first incubation. Then, after incubation for 10 minutes, a test solution containing fluorescein-labeled adenosine and monoclonal anti-adenosine antibodies is added. Residual adenosine and fluorescein-labeled adenosine compete for binding to the antibody. The amount of labeled adenosine bound to the antibodies is determined by a known fluorescence polarization method and the homocysteine concentration is determined by interpolation from a standard curve.

Example 5

Assay buffer: 0.1 M phosphate buffer (pH 7.4) containing 1 mg / ml rabbit IgG and 10 mM dithiothreitol.

SAH Hydrolase Solution: 40 milliliters / ml S-adenosyl-1 homocysteine hydrolase dissolved in assay buffer

Adenosine solution: 5-10 ^-8 M adenosine dissolved in assay buffer.

Adenosine deaminase solution: 200 milliliters / ml S-adenosyl-1-homocysteine hydrolase dissolved in assay buffer.

Phenol / Nitro Prussian solution. 10 mg / ml phenol and 50 pg / ml nitroprusside sodium dissolved in water.

Hypochlorite solution: 11 mM NaOCl in 125 mM NaOH.

Execution of the test

1. 75 [mu] l of adenosine solution and 75 [mu] l of SAH hydrolase solution are mixed with the test sample (preferably pre-treated according to Examples 1-4) and kept at 37 [deg.] C. for 10 minutes.

2. Add 100 µl of adenosine deaminase solution and keep at 5 ° C for 5 minutes.

3. Add 750 µL phenol / nitroprusside solution and 750 µL hypochlorite solution. After 30 minutes at 37 ° C, extinction was measured at 628 nm. In parallel, the assay was repeated but without the addition of SAHhydrolase. The difference between the two extinction values at 628 nm was determined and the homocysteine concentration was determined by interpolation from a standard curve using known standards.

Example 6

Preparation of fluorophore-labeled SAH A 10 mM SAH solution was prepared with dimethylformamide and diluted 1:10 in 100 mM sodium phosphate buffer, pH 7.5. To the resulting solution was added fluorescein isothiocyanate to a final concentration of 1 mM. After incubation at room temperature for 60 minutes, the SAH-fluorescein conjugate was purified by HPLC using a Chromasil C-18 column at 260 nm using a gradient of 25 mM ammonium acetate (pH 7.0) and methanol.

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Example 7

Development of anti-SAH antibodies

a) Preparation of antigen for SAH at a concentration of 125 mM

Bovine serum albumin is added to a final concentration of 5 mg / ml in 25 mM phosphate buffer (pH 7.4) containing NaCl. To the resulting mixture was added bis (succinimidyl) suberate to a final concentration of 1 mM and the reaction mixture was allowed to react for 60 minutes. (During the above coupling reaction, the pH is kept low, thereby facilitating coupling at the amino group of adenosine to that of the homocysteine.) Elution is carried out with a phosphate buffered saline solution using a Pharmacia Superose 12 column.

b) Development of hybridomas

The above antigen forms hybridomas with James

W. Gooding (Monoclonal Antibodies: Principle and Practice, Academic Press, London, 1983, Chapter 3).

c) Selection of hybridomas (i) Hybridomas producing anti-SAH antibodies are identified as follows. The IgG content of the supernatant of the hybridomas was measured by a known ELISA method. The supernatant is mixed in a cuvette with a buffer containing 50 mM phosphate, 120 mM sodium chloride and 0.1 mg / ml rabbit IgG, pH 7.4, to a final concentration of 0.1 pmol / l mouse IgG. Fluorescein-labeled SAH prepared according to Example 6 was added to a final concentration of 0.02 pmol / L. After incubation for 10 minutes, measure the degree of polarization with a spectrophotometer equipped with a fluorescence polarization unit

A = fluorescence intensity when the polarization plane of the incident light is parallel to the polarization plane of the filter used to filter the emitted light,

B = fluorescence intensity when the polarization plane of incident light is perpendicular to the polarization plane of the filter used to filter the emitted light using an excitation wavelength of 494 nm and detecting the emitted light at 517 nm.

The degree of polarization is calculated using the equation (A-B) / (A + B). The low degree of polarization indicates that the monoclonal mouse antibody does not bind to SAH.

(ii) From the hybridomas selected as described in (i), hybridomas which produce antibodies reactive with adenosine and / or homocysteine are identified as follows. Monoclonal anti-SAH antibodies from the hybridoma supernatant were coated on the surface of the wells of the microtiter plate as described in Example 9 below. ¹⁴ C-labeled adenosine (or ¹⁴ C-labeled homocysteine) in buffer containing 25 mM phosphate, 120 mM sodium chloride and 0.1 mg / ml rabbit IgG (pH 7.4) (manufactured by Amersham Ltd., UK) into microtiter wells. After incubation for 60 minutes, the wells are washed three times with the above buffer (which of course does not contain adenosine or homocysteine). The high level of radioactivity remaining in the wells indicates that the hybridomas themselves produce antibodies that bind to adenosine (or homocysteine). These antibodies are not used in the assay.

(iii) Production of monoclonal IgG. Hybridomas that produce antibodies that bind to SAH but not adenosine or homocysteine according to (i) above are selected for monoclonal IgG production. Selected hybridomas are used to induce ascites in mice or to produce cell cultures in vitro according to known methods. Monoclonal antibodies are isolated from ascites or cell culture media by known methods (see James W. Gooding, Monoclonal Antibodies: Principle and Practice, Academic Press, London, 1983).

Example 8

Immunoassay for fluorescence polarization of L-homocysteine

Enzyme solution: 0.2 mg / ml rabbit IgG, 120 mM

50 mM phosphate buffer, NaCl, 10 mM dithiothreitol, 10 units S-adenosyl-1-homocysteine hydrolase and 0.1 mM adenosine, pH

7.4) ·

Fluorescein-Labeled SAH Solution: The fluorescein-coupled SAH prepared in Example 6 was dissolved to a final concentration of 1 pmol / L in 50 mM phosphate buffer, pH 7.4 containing 125 mM NaCl and 0.2 mg. / ml rabbit IgG

Antibody Solution: Monoclonal anti-SAH antibodies (which do not react with adenosine, and preferably not with homocysteine), prepared as in Example 7, are dissolved in a final concentration of 0.1 pmol / L in 50 mM phosphate buffer, pH 7.4. ) containing 125 mM NaCl and 0.2 mg / ml rabbit IgG.

Execution of the test

In a cuvette, 15 μΐ of plasma (first known series of homocysteine-containing samples) is mixed with 100 μΐ of enzyme solution and kept at 37 ° C for 15 minutes. Add 100 μΐ of fluorescein-labeled SAH solution followed by 1.0 mL of antibody solution. The degree of polarization was measured using a spectrophotometer equipped with a fluorescence polarization unit as described in Example 7, step (c) (i) and plotted against homocysteine concentration.

The assay may also be performed using the labeled haptens and antibodies of Examples 11 and 13, or 15 and 17 instead of those of Examples 6 and 7.

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Example 9

Enzyme-linked immunoassay on microtiter plate (a) Enzyme solution

The enzyme solution of Example 8 was used.

(b) Preparation of a peroxidase-labeled SAH solution

Dissolve 0.5 mg of horseradish peroxidase in 1 ml of purified water. 200 µl of 0.02 M sodium periodate solution was added and the mixture was stirred at room temperature for 20 minutes and dialyzed against 10 mM sodium acetate buffer, pH 4.4 overnight. SAH was added to a final concentration of 0.1 mM and the pH was adjusted to 6.0. The solution was stirred at room temperature for 4 hours. 100 μΐ of freshly prepared 4 mg / ml aqueous sodium borohydride solution was added and the reaction was incubated at 4 ° C for 2 hours. Peroxidase and its SAH conjugates were isolated by size exclusion chromatography on a Superose 6 column (Pharmacia, Sweden).

(c) Coating microtitering wells with anti-SAH antibodies

Sheep polyclonal sheep IgG from sheep immunized against mouse IgG was dissolved at a final concentration of 1 mg / ml in 100 mM borate buffer, pH 9.0. 300 μΐ of the above solution was filled into each well of a polystyrene microtiter plate. After incubation at 37 ° C for 120 minutes, the wells are washed five times with phosphate buffered saline. Thereafter, the monoclonal mouse IgG anti-SAH antibodies prepared according to Example 7 were dissolved in a final concentration of 50 pg / ml in phosphate buffered saline. 200 μΐ of the resulting monoclonal IgG solution was added to each well and incubated at 37 ° C for 120 minutes. The wells are then washed five times with phosphate buffered saline containing 0.1 mg / ml rabbit IgG.

(d) Assay: Plasma samples (first a series of samples of known homocysteine concentration) are mixed with 500 µl of enzyme solution and kept at 37 ° C for 15 minutes. 50 µl of the peroxidase-labeled SAH solution was added, mixed, and 500 μ a of the resulting mixture was added to each well of the anti-SAH antibody-coated microtiter buffer as in step (c) above and buffered to pH 7.4. After incubation at 37 ° C for 60 minutes, the wells were washed three times with phosphate buffered saline containing 0.1 mg / ml rabbit IgG. Add 100 μΐ of a solution of orthophenylenediamine in 0.1 μl citrate buffer (pH 6.0) containing 0.015% hydrogen peroxide (100 μΐ) to each well. After 10-30 minutes, light absorbance was measured at 450 nm in each well. Absorption is plotted versus homocysteine concentration.

Example 10

Production of hapten

3-S- (1-Anhydro-D-ribofuranosyl) -thiopropylamine (Compound 1-1) (a) Activated Hydroxyl-Protected Mercaptan [In Formula (8): R] R.2 = H] is reacted with methyl pD-ribofuranoside (commercially available compound)] in a mixture of 5 equivalents of trimethylsilyl methanesulfonate and 1 equivalent of boron trifluoride etherate according to the method of Jun et al., Carb. Gap. 163: 247-261 (1987). 0.5 equivalents of 1-anhydro-D-ribose (compound 2) are added in fine powder, under small stirring, to the acetone / sulfuric acid mixture, which is prepared by slowly adding 6.3 mL of concentrated sulfuric acid in an ice bath. 100 ml of freshly distilled acetone. The ice bath was removed and the reaction was allowed to proceed at room temperature for 8 hours. The resulting white crystalline solid was dissolved in chloroform, washed with aqueous sodium hydroxide solution, dilute hydrochloric acid, and finally with water, dried and evaporated. The 2,3-isopropylidene derivative of 1-anhydro-D-ribose (5) is obtained. One equivalent of this and two equivalents of carbon tetrabromide are dissolved in anhydrous diethyl ether and cooled on ice. While stirring, 2 equivalents of triphenylphosphine are slowly added. The ice bath was removed and the mixture was allowed to warm to room temperature. A slight evolution of hydrogen bromide was observed during the reaction. After completion of the reaction, methanol was added to the reaction mixture. The bromide salt (6) is isolated by filtration and evaporation of the filtrate. To the bromide derivative was added 1 equivalent of thiourea dissolved in warm water and diluted with rectified alcohol. The mixture is refluxed and shaken occasionally, and the reaction is continued for about 30 minutes after dissolution of the bromide derivative. The reaction mixture was cooled on ice and filtered. The resulting solid is treated with alkaline water to afford the hydroxyl protected mercaptan (7) in the organic phase. This is converted to the active form (8) by treatment with methoxide in methanol. The resulting compound is reacted as follows.

(b) 3- [S- (1-Anhydro-D-ribofuranosyl) thio] propylamine equivalent The compound prepared according to Example 10 (a) (Compound 8) is reacted with 1 equivalent acrylonitrile to give a thioether [( compound 9)] is obtained, which is reduced by treatment with ₄ LíA1H del anhydrous diethyl ether, and isolating the free amine by treatment with [compound (10)] with aqueous hydrochloric acid deprotected.

The corresponding aminopropylthioethers, wherein R 1 and R ₂ are other than hydrogen, are prepared analogously using, for example, commercially available compounds of formulas (1) and (2).

Example 77

Production of labeled hapten

A solution of the compound of Example 10 in dimethylformamide (50 mM) was diluted 1: 5 with 0.1M hydrogen carbonate (pH 9.2). To the resulting solution was added fluorescein isothiocyanate to a final concentration of 12 mM. After incubation at room temperature for 60 min, the fluorescein conjugate of Example 10 was purified by RPC, Kromasil

HU 219 607 Β

100 C C-18 column and gradient mixture of 20 mM ammonium acetate (pH 7.0) and methanol.

Example 12

Production of antigen

To a solution of the compound of Example 10 in 50 mM phosphate and 125 mM NaCl, pH 7.4, was added bovine serum albumin (BSA) to a final concentration of 5 mg / ml. To the resulting mixture was added bis (sulfosuccinimidyl) suberate to a final concentration of 1.2 mM and the reaction was allowed to react for 60 minutes. The protein-like fraction, which also contains the hapten-BSA conjugate, was purified by size exclusion chromatography using a Pharmacia Superose 12 column and phosphate buffered saline as eluent.

Example 13

Preparation of antibody

Antibodies to the compound of Example 10 were prepared analogously to the procedure of Example 7 using the antigen of Example 12. Antibodies that do not react with SAH and react with adenosine are not used, since antibodies reactive with homocysteine are preferred.

Example 14

Production of hapten

N-hydroxysuccinimidyl-3- [S- (1-anhydro-D-ribofuranosyl) thio] butanoate [R 13 = R ₂ = HJ equivalent in Example 13 (a) freshly The resulting compound is reacted with 1 equivalent of ethyl 4-bromobutyrate to give the ester 11. This is hydrolyzed to the free acid by alkaline hydrolysis using aqueous sodium hydroxide solution in dioxane and deprotected by treatment with aqueous hydrochloric acid to give the free acid 12. 1 equivalent of this is mixed with 2 equivalents of N-hydroxysuccinimide in ice cold dimethylformamide and 1.2 equivalents of dicyclohexylcarbodiimide are added with constant stirring. The reaction was allowed to proceed at room temperature for 18 hours, after which the mixture was cooled and ice-cold diethyl ether was added. The precipitated NHS ester (13) is recrystallized from dimethylformamide / diethyl ether, dried and stored in a desiccator at 4 ° C.

The corresponding NHS esters wherein R 1 and R ₂ are other than hydrogen are prepared analogously using commercially available compounds of formulas (1) and (3).

Example 75

Production of labeled hapten

A solution of the compound of Example 14 in dimethylformamide (50 mM) was diluted 1: 5 in 0.1M hydrogen carbonate buffer (pH 9.2). To the resulting solution is added 5-aminoacetamido-fluorescein (fluoresceinylglycinamide) to a final concentration of 12 mM. After incubation at room temperature for 60 minutes, the fluorescein conjugate of 3- [S- (1-anhydro-D-ribofuranosyl) thio] butanoic acid was purified by RPC from a Kromasil 100 A C-18 column and 20 mM ammonium acetate. (pH 7.0) and a gradient of methanol.

Example 16

Preparation of antigen in mg / l, 50 mM phosphate and

To the BSA solution in 125 mM NaCl buffer (pH 7.4) is added the final compound of Example 14 to a final concentration of 1 mM. The reaction mixture is allowed to react for 60 minutes and the protein fraction, which also contains the BSA-hapten conjugate, is purified by size exclusion chromatography using a Pharmacia Superose 12 column and phosphate buffered saline as eluent.

Example 7

Preparation of antibody

Antibodies to Example 14 were prepared in an analogous manner to Example 7 using the antigen of Example 16. Antibodies that do not react with SAH and react with adenosine are not used, since antibodies reactive with homocysteine are preferred.

Example 18

Fluorescence Polarization Immunoassay Enzyme solution: 4 mg / ml casein, 120 mmol / l

50 mM phosphate buffer, pH 7.4 containing NaCl and 10 units / l S-adenosyl-1 homocysteine hydrolase.

Dithiothreitol (DTT) solution: Dithiothreitol was dissolved in water at a concentration of 50 mM and adjusted to pH 3.0 with HCl.

Adenosine solution: 1.8 mM adenosine dissolved in 50 mM phosphate buffer.

Fluorescein Labeled SAH Solution: A 50 mM phosphate buffer, pH 7.4, containing fluorescein coupled SAH prepared in Example 6.

Antibody Solution: Monoclonal anti-SAH antibodies (prepared as in Example 7) were dissolved in a final concentration of 0.1 pmol / L in 50 mM phosphate buffer, pH 7 containing 120 mM NaCl and 1 mg / ml casein. 4).

Execution of the test

1st step:

In a cuvette, 15 μΐ sample, 10 μΐ enzyme solution and μΐ adenosine solution are mixed with 10 μΐ acid DTT and kept at 37 ° C for 15 minutes.

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Step 2:

To the contents of the cuvette, add 100 μΐ of fluorescein-labeled SAH solution followed by 1.0 ml of antibody solution. The degree of polarization is measured using a spectrophotometer equipped with a fluorescence polarization unit as described in Example 7, step (c) (i) and plotted against homocysteine concentration.

Example 19

Fluorescence Polarization Immunoassay Enzyme solution: 4 mg / ml casein, 120 mmol / l

50 mM phosphate buffer, pH 7.4 containing NaCl and 10 units / l S-adenosyl-1 homocysteine hydrolase.

Dithiothreitol (DTT) solution: Dithiothreitol was dissolved in water at a concentration of 50 mM and adjusted to pH 3.0 with HCl.

Fluorescein-labeled SAH / adenosine solution:

pmol / l 50 mM phosphate buffer, pH 7.4, containing fluorescein-coupled SAH and 1.8 mM adenosine.

Antibody Solution: Monoclonal anti-SAH antibodies (prepared as in Example 7) were dissolved in a final concentration of 0.1 pmol / L in 50 mM phosphate buffer containing 120 mM NaCl and 1 mg / ml casein, pH

7.4).

Execution of the test

In a cuvette, 10 μΐ of plasma, 100 μΐ of enzyme solution and 10 μΐ of labeled SAH / adenosine solution are mixed with 30 μΐ of acid DTT and kept at 37 ° C for 15 minutes.

After completion of the incubation, 1.0 ml of antibody solution is added. The degree of polarization is measured using a spectrophotometer equipped with a fluorescence polarization unit as described in Example 7, step (c) (i) and plotted against homocysteine concentration.

The assays of Examples 18 and 19 can also be performed using the labeled haptens and antibodies of Examples 11 and 13 or 15 and 17 instead of the ones prepared in Examples 6 and 7.

Example 20

Luminescence examination

Assay buffer I: 1 mg / ml casein, 10 mmol / l DTT,

50 mM Pipes buffer pH 6.6 containing 0.5 mM MgCl ₂ and 30 mM KCl.

II. assay buffer: 4 mM EDTA, 20 mM

Hepes buffer (pH 7.75) containing 40 mM MgCl ₂ and 0.36 mM DTT.

III. assay buffer: 1.6 pg / ml luciferase (from Photinus pyralis), 700 pmol / L D-luciferin, 20 mM MgCl ₂ , 4 mM EDTA, 0.36 mM DTT and Hepes buffer, pH 7.75, containing 0.3 mM AMP.

Execution of the test

To 3 µl of assay buffer I was added 3 units of S-adenosylhomocysteine hydrolase, 20 µl of assay sample and incubated for 15 minutes at 37 ° C. Adenosine dissolved in assay buffer I was then added to a final concentration of 5 x 10 ^-6 mol / l. After incubation for 5 minutes, 750 µl containing 0.7 · 10 ^-5 M ATP and 1 milliliter adenosine kinase

Assay buffer I was added and the resulting solution was incubated at 37 ° C for an additional 5 minutes. The solution was diluted 1: 100 in volume with II. assay buffer and immediately add 500 μΐ of the diluted solution to 500 μΐ III. assay buffer. AII. and III. assay buffer was also equilibrated at room temperature (21 ° C). The resulting luminescence is measured at 550 nm with a photometer.

A parallel assay is performed without S-adenosyl homocysteine hydrolase. In clinical studies, homocysteine concentrations are calculated by interpolating the difference between S-adenosyl homocysteine hydrolase and standard values.

Example 21

Preparation of polyclonal antibodies

Rabbit polyclonal antibodies to the antigens of Examples 12 and 16 are produced according to the protocol published by Dako Corporation (Copenhagen, Denmark). Polyclonal IgG is purified from the collected antiserum according to the same protocol. Polyclonal antibodies are purified from antibodies reacting with adenosine and homocysteine residues by passing the antibodies on a Racti-Gel column containing immobilized adenosine and homocysteine residues (manufactured by Pierce Chemical Company, Belgium) according to the manufacturer's protocol. Antibodies that do not react with SAH are also not used. Selected antibodies can be used in the assays described in the examples above.

In the methods described below for homocysteine (Hcy) assays, the disulfide bonds between homocysteine and other compounds are cleaved by incubation with a reducing agent at 37 ° C for 5 to 60 minutes in buffer. A suitable reducing agent is dithiothreitol (DTT) at a final concentration of 3 mM.

The following enzymes were used to determine homocysteine: Betaine-Homocysteine Methyltransferase Dimethyltetin-Homocysteine Methyltransferase Methionine Synthetase Homocysteine Cystation-β-Synthetase.

Example 22

Determination of homocysteine using betaine homocysteine methyltransferase

homocysteine + betaine-> methionine + dimethylglycine betaine-homocysteine methyltransferase

HU 219 607 Β methionine + ATP-> S-adenosylmethionine + methionine ade- + pyrophosphate nosyl synthetase

According to Oden et al., Biochemistry 22: 2978-2986 (1983), the enzyme methionine adenosyl synthase is present in the cytosol of human erythrocytes. The cytosol preparation is prepared as follows.

1. Preparation of Methionone Adenosyl Synthetase Volume of blood (5 ml) was taken from volunteers using Liheparin as anticoagulant and centrifuged at 1000 g for 10 minutes. The supernatant (platelet rich plasma) and the supernatant of white blood cells are removed by pipette. The remaining 3 ml of erythrocyte was transferred to a larger centrifuge tube and 30 ml of ice-cold wash (50 mM sodium phosphate buffer, pH 7.4, 50 mM NaCl) was added, mixed gently and with 2500 g. Centrifuge at 12 ° C for 12 minutes. The supernatant was removed, additional 30 mL of detergent was added and the centrifugation and resuspension was repeated three times. The final pellet containing the washed erythrocytes is resuspended in 2x5 ml of ice-cold water and incubated on ice for 2 hours. The suspension is then centrifuged at 20,000 g for 30 minutes in a SW55T1 rotor to remove cell membranes and debris. The supernatant was dialyzed overnight at 4 ° C against 3 L of 30 mM KCl - 40 mM HEPES buffer, pH 7.4. The final supernatant was collected and used for further assays.

2. Preparation of the reaction mixture

120 μΐ 10 mM ATP - 2 mM Beta - 30 mM MgCl ₂ - 26 mM KCl - 35 mM HEPES buffer (pH 7.5)

110 μΐ erythrocitalate μΐ Betaine homocysteine methyl transferase solution (2250 units / ml) μΐ serum sample, 0-25-50 and 100 pmol / l homocysteine added

DTT was added to a final concentration of 3 mM. After incubation at 37 ° C for 2, 5 and 24 hours, the reaction mixture was analyzed by competitive immunoassay for S-adenosylmethionine using a monoclonal antibody against S-adenosyl homocysteine (SAH), which shows 20% cross-reactivity to S-adenosyl methionine. . Assays were performed on a BSA-S-adenosyl-L-homocysteine-coated microtiter plate. Incubate 25 μΐ of each reaction mixture with 200 μΐ of antibody solution in 100 mM sodium phosphate buffer, pH 7.4 at 20 ° C for 20 minutes. The fluid in the wells was removed and the wells were washed three times with the latter buffer and incubated with rabbit anti-mouse antibody and horseradish peroxidase (HRP) conjugate for 20 minutes. After washing, 200 μΐ of substrate (tetramethylbenzidine) was added to the wells, and the microtiter plates were incubated for 15 minutes and the reaction was stopped by the addition of 0.8 M sulfuric acid. The color is read at 450 nm in a microtiter plate reader. From the results, a dose-response curve for the added homocysteine is shown in Figure 1 after 24 hours of incubation.

The experiment was repeated with the ATP concentration reduced to 0.1 mmol / l. This resulted in a lower background value. The dose-response curve obtained after 3 hours of incubation is shown in Figure 2.

Methionine produced by the conversion of homocysteine can also be determined microbiologically (see Example 27).

Example 23

Assay of homocysteine using dimethyltetin homocysteine methyltransferase

homocysteine + dimethyltetin-> methyltetin + methionine dimethyltetin homocysteine methyltransferase methionine + ATP-> S-adenosylmethionine + pyrophosphate methionine adenosyltransferase

Dimethyltethin and dimethyltin homocysteine methyltransferase enzyme are added to the serum sample and incubated. The amount of methionine produced is determined by evaluating the growth rate of methionine dependent bacteria, by fluorescence assay, or by immunoassay.

Experimental method

1. For a 100 μΐ buffer solution containing a reducing agent (3 mM DTT or TCEP, 100 mM sodium phosphate, 0.15 mM NaCl, pH 7-9, depending on the reducing agent used), 50 μΐ Hcy sample or calibrators ( Hcy of known concentration is added and the mixture is incubated at 37 ° C for 10 minutes.

2. At the end of the incubation, 50 μΐ buffer (50 units enzyme, 50 mM sodium phosphate, 0.15 M NaCl, pH 7.5) containing dimethyltin homocysteine methyltransferase is added. The mixture was incubated at 37 ° C for 30 minutes (free Hcy (i.e., reduced Hcy) is methylated to L-methionine).

3. After incubation step 2, a 50 μΐ solution of methionine adenosyltransferase (50 units enzyme in 50 mM sodium phosphate, 0.15 M NaCl, pH 7.5 buffer containing 100 pmol / L ATP, Containing 0.5 mM KCl and 0.5 mM MgCl) and the resulting solution was incubated at 37 ° C for 30 minutes [Met transforms to S-adenosyl-L-methionine (SAM) ].

4. The amount of SAM formed in Step 3 is determined by enzyme immunoassay on a microtiter plate as follows:

(a) After incubation, 25 μΐ of the sample is transferred to wells coated with bovine serum albumin-SAM conjugate (BSA-SAM) on the microtiter plate.

b) 200 μΐ of mouse anti-SAM antibody (approximately 100 pg / ml, 100 mM sodium phosphate, 0.15 M NaCl, pH 7.5) in 0.1 mg / ml is added to each well. bovine γ-globulin, 2 mg / ml BSA and 0.5 ml / l Tween 20] ("assay buffer"), and

Incubate at room temperature (18-25 ° C) for 30 minutes.

(c) Wash wells three times with 400 μΐ wash solution (1:10 diluted assay buffer) and add 200 μΐ HRP-conjugated anti-mouse antibody (approximately 1.5 pg / ml in 'stabilizer buffer'). The wells were incubated at room temperature for 30 minutes and then washed three times with 400 μΐ wash solution.

d) 200 μΐ HRP substrate was added to each well and incubated for 10 minutes at room temperature.

(e) The HRP reaction was stopped by the addition of 100 μΐ 0.8 M sulfuric acid and the resulting yellow color was measured at 450 nm.

Calibration method

Calibrators used in the assay are prepared by crystallizing L-monocystine in 10 mM sodium phosphate, 0.15 M NaCl, pH 7.5, at known concentrations (up to 50 pmol / l, e.g. 8, 20, 30 and 50 pmol / L). Plot the concentration (log ₁₀ values) of the Hcal calibrators against the measured absorbance values and construct the calibration curve using a 4 parameter logistic equation. The resulting curve is then used to determine the Hcy values of the unknown samples based on the absorbance measurement.

Samples containing more than 50 pmol / L of free Hcy are diluted with buffer (10 mM sodium phosphate, 0.15 M NaCl, pH 7.5) prior to assay.

Example 24

Investigation of homocysteine by methionine synthase

Principle of examination:

homocysteine + N ⁵ - methionine + tetrahydrofolate ^> + tetrahydrofolate methionine synthetase Vitamin B ₂

N ⁵ -tetrahydrofolate, methionine synthase enzyme and vitamin B ₁₂ (cobalamin) were added to the serum sample and incubated. The amount of methionine formed is determined by measuring the growth rate of methionine-dependent bacteria, fluorescence, or immunoassay.

The reaction mixture was 500 pmol / lN ⁵ -methyl tetrahydrofolate, 50 pmol / l cyanocobalamin, 300 pmol / l S-adenosylmethionine, 125 mmol / 12-mercaptoethanol, methionine synthetase and 50 mmol / l potassium. phosphate buffer (pH

7.4), the mixture is incubated for approximately 20 minutes at 37 ° C to completely convert the homocysteine to methionine.

Example 25

Examination of homocysteine by homocysteine

Principle of examination:

homocysteine-2-keto-butyric acid + NH ₃ + H ₂ S homocysteine

Testing:

1. Conversion of homocysteine to homocysteine and measurement of H ₂ S formed by homocysteine> 2-keto-butyric acid + NH ₃ + H ₂ S homocysteine

K ₃ Fe (CN) ₆

H ₂ S + N, N-dibutyl-> 3,7- (N, N-dibutylamino) 1,4-phenyldiamine phenothiazine

procedure:

1. Plasma samples are obtained by collecting the blood in a 0.11 M sodium citrate solution and centrifuging. Four aliquots were taken from a plasma sample containing 9 pmol / l homocysteine and 25, 50, 75 and 100 pmol / l purified homocysteine was added to the aliquots.

2. Two 0.1 mL aliquots of each sample are added to 0.9 mL of 1 mM dithiothreitol, 0.2 mM sodium citrate, 2% Triton Χ-100, 40 mM containing sodium borate buffer, pH 9.0. The mixtures were incubated at 37 ° C for 60 minutes to release any homocysteine which is attached to other compounds via a disulfide bond.

3. Add 20 µl of a 10 mM potassium phosphate buffer (pH 7.6) solution containing 0.06 mg / ml homocysteinase enzyme to one of each pair of incubation mixtures and incubate for exactly 10 minutes at 37 ° C. -you. This results in the enzyme-treated mixture through the two-homocysteine ketovaj acid, ammonia and H ₂ S into cDNA, while the untreated mixtures form part of the blank.

4.40 µl of 14.65 mg / ml N, N-dibutyl-1,4-phenyl diamine hydrochloride in 6 mol / l HCl and 20 µl of 10 mM potassium phosphate buffer, pH 7, 6) 100 mM K ₃ Fe (CN) ₆ solution was added simultaneously and the solution was stirred rapidly. The H ₂ S produced by the enzyme then produces a colored phenothiazine derivative. After incubation at 37 ° C for 10 minutes, the absorbance of each reaction mixture was measured at 675 nm in a spectrophotometer and blank results were subtracted from those obtained with enzyme-treated samples. The following results are obtained.

Concentration of homocysteine Absorption 675 pmol / l 25 0,036 50 0,084 75 0,130 100 0,170

A graphical representation of the results (Figure 3) shows that there is a dose-effect relationship, so the assay is suitable for the determination of homocysteine in plasma.

2. Conversion of homocysteine to homocysteine and measurement of the formed NH ₃ by homocysteine> 2-keto-butyric acid + NH ₃ + H ₂ S homocysteine

Na ₂ (Fe (CN) ₅ NO), NaOCl

NH ₃ + phenol → indophenol blue compound

process

1. Plasma samples are obtained by collecting blood in a 0.11 mol / l sodium citrate solution

And centrifuge. Four aliquots were taken from a plasma sample containing 9 pmol / l homocysteine and 25, 50, 75 and 100 pmol / l purified homocysteine was added to the aliquots.

2. Two 0.1 mL aliquots of each sample are added to 0.9 mL of 1 mM dithiothreitol, 0.2 mM sodium citrate, 2% Triton Χ-100, 40 mM containing sodium borate buffer, pH 9.0. The mixtures were incubated at 37 ° C for 60 minutes to release any homocysteine which is attached to other compounds via a disulfide bond.

3. Add 20 µl of a 10 mM potassium phosphate buffer (pH 7.6) solution containing 0.06 mg / ml homocysteinase enzyme to one of each pair of incubation mixtures and incubate for exactly 10 minutes at 37 ° C. -you. Homocysteine is then converted to 2-keto-butyric acid, ammonia and H ₂ S in the enzyme-treated mixtures, whereas the untreated mixtures are the blank.

4. The incubation mixtures contained 350 each containing 350 pl of 50 mmol / 1 NaOCl to 125 mmol / 1 NaOH and 350 pl eg 100 mmol / 1 of phenol and 0.17 mmol / 1 of Na ₂ Fe (CN) ₅ Mix with a solution containing NO (nitro-prusside sodium) and incubate at about 20 ° C for 45 minutes. Samples containing NH _{3 give a} blue indophenyl compound. Each enzyme-treated mixture was measured in a spectrophotometer at 628 nm against the appropriate blank.

A graphical representation of the results (Figure 4) shows that there is a dose-effect relationship and is therefore suitable for assaying homocysteine in plasma.

Depending on the concentration in the homocysteine sample, the amount of incubation mixture in step 4 may be varied from 50 to 350 µl, so that the incubation period may vary from 15 to 45 minutes.

Example 26

Determination of homocysteine by cystathionine β-synthetase

Principle of examination:

homocysteine + serine> cystathionine cystathionine β-synthetase

The cystathionine β-synthetase enzyme is added to the serum sample and incubated, and the amount of cystathionine produced is determined by one of the following methods.

a) Cystathionin → homoserine + cysteine cystathionine transsulfurase

The cystathionine transsulfurase enzyme is added to the above reaction mixture, incubated and the amount of cysteine formed is measured.

(b) cystathionine-2-keto-butyric acid + cysteine cystathionine lyase,

Vitamin B ₆

The cystathionine lyase enzyme and vitamin B ₆ (pyridoxal phosphate) added to the above reaction mixture, incubated, and then measuring the amount of formed 2-ketovajsav or cysteine.

Assay: determination of cysteine

A 0.5 ml sample containing cysteine from the conversion of homocysteine obtained by one of the above methods is mixed with 0.5 ml glacial acetic acid and 0.5 ml solution containing 25 mg / ml ninhydrin, 60% acetic acid and 0.24 M phosphoric acid. The mixture was heated in a hot water bath for 10 minutes and cooled to about 20 ° C. The contents of each reaction mixture were diluted to 5 ml with 95% ethanol and mixed. Absorption was measured at 500 nm in a spectrophotometer against a suitable blank in which one or the other of the homocysteine converting enzymes was omitted from the initial reaction mixture. The molar amount of cysteine is determined from a standard curve and corresponds directly to the molar amount of homocysteine originally present in the serum sample.

Assay: Determination of 2-keto-butyric acid

a) Use of ketobutyrate dehydrogenase

To the resulting reaction mixture containing 2-keto-butyric acid is added NADH together with the enzyme ketobutyrate dehydrogenase to catalyze the following reaction:

2-Ketobutyrate + 2-Hydroxybutyrate + + NADH * + NADH Ketobutyrate Dehydrogenase

The result is read as a decrease in absorbance at 340 nm, which is due to a decrease in NADH.

b) Use of propionyl CoA synthetase

To the resulting reaction mixture containing 2-keto-butyric acid is added NAD, together with the propionyl CoA synthetase enzyme, which catalyzes the following reaction:

2-Keto Butyric Acid + Propionyl CoA + + NAD + CoASH * + NAD Propionyl CoA Synthetase

The result is measured as the increase in absorbance due to the formation of NADH at 340 nm.

Example 27

Microbiological determination of methionine (applicable to the assays of Examples 22-24)

E. coli 15-3 (pro-22, met F63) in 50 ml

Vogel-Bonner (VB) was cultured in minimal medium containing 0.2% glucose, 50 pg / ml proline, 50 pg / ml methionine and 0.001% thiamine. Cells are cultured at 600 nm to an absorbance of about 1.0, and the cells are harvested by centrifugation at 5000 g for 5 minutes. The methionine-containing supernatant was discarded and the cells were resuspended in 50 ml of VB medium and centrifuged for two cycles. In the final wash, the cells are resuspended in 5 ml of VB medium containing 20% glycerol and stored frozen at -80 ° C until the microbiological determination of methionine.

The reaction mixtures (in which methionine is formed from homocysteine) are kept in a hot water bath for 5 minutes. Each reaction mixture was then transferred to centrifuge tubes and heated at 12,000 g for 15 minutes.

HU 219 607 HU centrifuged to settle denatured proteins. The supernatant is then used to test for methionine.

Microbiological examination of methionine was performed on a microtiter plate, whereby 200 µl of minimal medium containing 5 x 10 ⁶ colony forming units of E. coli 15-3 was added to each well. The culture medium contains VB salts, 0.2% glucose, 50 pg / ml L-proline and 0.001% thiamine.

Supernatant supernatants from the enzyme reaction treated as above are added to the wells in a total volume of 100 µl, after further dilution with optional distilled water, depending on the concentration of methionine. The blank was prepared from a reaction mixture in which an enzyme or a co-substrate essential for converting homocysteine to methionine was omitted.

The microtiter plates were incubated at 37 ° C for 30 hours. The cells were resuspended and the absorbance was measured at 450 nm in each well. The absorbance reduced by the blank absorbance is directly proportional to the amount of methionine formed in the reaction mixture. The exact concentration can be determined from a standard curve obtained by using samples containing known concentrations of methionine instead of the supernatants obtained in the enzymatic reaction.

Example 28

Fluorescence assay for methionine (applicable to the assays described in Examples 22-24) 100 μΐ of the methionine-containing mixture formed in any of the enzyme reactions described above was purged with nitrogen and added with 50 μΐ of bis (3,5,5-trimethylcyclohexyl) phthalate After incubation at 37 ° C in the dark for 10-30 minutes, the reaction was stopped by the addition of 10 μΐ 4 M perchloric acid. After 1 minute, the acid was neutralized by addition of a solution containing 10 μΐ of 4 M KOH and 3.3 mM potassium bicarbonate, and the precipitate was removed by centrifugation.

O-phthalaldialdehyde (OPA) reagent was prepared by dissolving OPA in methanol (56 mM) and 1 ml of the resulting solution in 9 ml of 0.1 M sodium borate buffer, pH 9, 5) and 40 μΐ 2 mercaptoethanol.

μΐ of the above neutralized reaction mixture was mixed with 175 μΐ of OPA reagent. After 2 minutes at 23 ° C, 220 μΐ of the mixture was injected onto an ODS Hypersil column equilibrated in 50 mM sodium phosphate buffer (pH 5.0) containing 30% methanol. The columns were then eluted with a linear gradient of 30-55% methanol / phosphate buffer (0-12 min) followed by a linear gradient of 55-80% methanol / phosphate buffer (12-15 min) at a flow rate of 2 ml / min. The adducts of o-phthalaldehyde with methionine and norvaline show retention times of 13.5 and 15.5 minutes, respectively. The methionine peak is integrated and quantified against a standard curve.

Example 29

Determination of methionine by enzymatic method

The determination of methionine can also be performed by enzymatic conversion of methionine as follows. Methionine + ATP-> S-adenosylmethionine + inorganic pyrophosphate methionine adenosyltransferase

22-24. ATP and methionone adenosyltransferase enzyme are added to the reaction mixture according to any one of Examples 1 to 4 and the following components are measured:

- Loss of ATP

- formation of pyrophosphate

- Formation of S-adenosyl methionine

Definition of TP

A commercially available kit, such as the ATP bioluminescence assay kit manufactured by Boehringer Mannheim, can be used to determine ATP depletion, i.e., ATP is determined in a blank and compared to a complete sample / reagent mixture. The assay is based on the luciferase enzyme which catalyzes the following reaction.

ATP + luciferin> luciferyl adenylate + + inorganic pyrophosphate luciferase

The luciferyl adenylate is then converted to luciferin and AMP by oxygen, which is accompanied by light emission (bioluminescence) and can be determined by a luminometer or spectrofluorometer.

Determination of inorganic pyrophosphate

Inorganic pyrophosphate (PPi) can be measured by the following enzymatic transformations.

PPi + Glucose -> Glucose-6-Phosphate + Pi Glucose-6-Phosphatase

Glucose-6-phosphate + 6-phosphogluconolactone + + NADP * + NADP glucose-6-phosphate dehydrogenase

Thus, addition of glucose, NADP and the enzymes glucose-6-phosphatase and glucose-6-phosphate dehydrogenase to the reaction mixture in which the inorganic pyrophosphate is formed produces NADPH, which can be determined at 340 nm.

Determination of S-adenosyl methionine

The Axis antibody to S-adenosyl homocysteine gives a cross-reaction with S-adenosyl methionine and thus can be used in a competitive reaction with immobilized S-adenosyl homocysteine or immobilized S-adenosyl methionine to measure the amount of free S-adenosyl methionine formed.

Example 30

Determination of tetrahydrofolate (applicable to the assay of Example 24)

In the assay of Example 24, instead of measuring methionine, the tetrahydrofolate can be converted into a cyclical reaction to give

GB 219,607 Β nyeként formed N ^{5-methyl-tetrahydrofolate,} and simultaneously formed with one molecule of NADH is consumed in each molecule of homocysteine to methionine. Tetrahydrofolate + N ⁵ , N '° -methylene-tetra + serine * Hydrofolate + + Glycine

swimmers:

tetrahydrofolate 5,10hidroxi-methyltransferase

N ⁵ , N ¹⁰ -methylene- N ⁵ -tetrahydrofolate + tetrahydrofolate -> + NAD + NADH

N ⁵ J ⁴ O ¹⁰ -methylene tetrahydrofolate dehydrogenase

To the reaction mixture of Example 24 was added NADH and the enzymes Lerine: tetrahydrofolate-5,10-hydroxymethyltransferase and N ⁵ , N ¹⁰ -methyl-tetrahydrofolate dehydrogenase. We appreciate the reduction of NADH at 340 nm in a spectrophotometer.

Claims (38)

A method for determining homocysteine in a sample, comprising contacting the sample with a homocysteine converting enzyme and at least one substrate other than homocysteine of the enzyme, and determining the homocysteine co-substrate and the homocysteine co-substrate without chromatographic separation of the reagents or reaction products. unlabeled substance to be selected from the reaction products of its conversion, The method of claim 1, wherein the sample is contacted with an antibody against the substance to be determined and an hapten of the antibody other than the unlabeled substance to be determined, and the determination of the substance to be determined indirectly by determining the antibody bound or not. execute. The process according to claim 2, wherein the hapten is a polyhaptene. 4. The method of claim 2, wherein said hapten is a labeled molecule having an epitope structural unit that is also present in the unlabeled substance to be determined. 5. The method of any one of claims 1 to 3, wherein the antibody is a monoclonal antibody. The method of any one of claims 2-5, wherein the antibody is a matrix bound antibody. 7. The method of claim 1, further comprising contacting the sample with a second enzyme for converting the substance to be determined and determining the substance to be determined indirectly, either by using a substrate of the second enzyme other than the substance to be determined, or enzymatic conversion of a substance to be determined by the determination of a product of a second enzyme. 8. A method according to any one of claims 1 to 4, characterized in that the substance to be determined is the above-described co-substrate. 9. The method of claim 8, wherein the enzyme is S-adenosyl homocysteine hydrolase and the substance to be determined is adenosine. 10. The method of claim 8, further comprising contacting the sample with an adenosine-converting enzyme other than the homocysteine-converting enzyme, the substance to be assayed and, indirectly, the determination of adenosine, a co-substrate or adenosine-converting adenosine. conversion by enzyme assay. 11. The method of claim 10, wherein the adenosine converting enzyme is adenosine kinase. 12. The method of claim 10, wherein the adenosine converting enzyme is adenosine deaminase. 13. A process according to any one of claims 1 to 5, characterized in that the substance to be determined is the reaction product of the conversion. 14. The method of claim 13, wherein the enzyme is S-adenosyl homocysteine hydrolase and the substance to be determined is S-adenosyl homocysteine. 15. A method according to any one of claims 1 to 6, characterized in that the sample is a blood, plasma or urine sample pretreated with a disulfide bond cleavage reducing agent. 16. A method according to any one of claims 1 to 3, characterized in that the determination of the substance to be determined is carried out photometrically. 17. The method of claim 16, wherein the determination is performed spectrophotometrically or colorimetrically. 18. The method of claim 16, wherein the determination is carried out by turbidimetry or nephelometry. 19. The method of claim 16, wherein the determination is by fluorescence polarization detection. 20. 1-7, 13-17. A method according to any one of claims 1 to 6 and 19, wherein the determination of the substance to be determined is carried out indirectly by detecting the chromophore or fluorophore on the labeled S-adenosyl-homocysteine or labeled furanos-6-thioether. 21. The method of claim 1, wherein the blood, plasma or urine sample is treated with a reducing agent; contacting the sample with S-adenosyl homocysteine hydrolase; incubating the resulting mixture, then contacting it with ATP and adenosine kinase and incubating the mixture for at least 1 minute; contacting the resulting mixture with luciferin and a luciferase, and detecting the light generated. 22. The method of claim 1, wherein the blood, plasma or urine sample is treated with a reducing agent; contacting the sample with S-adenosyl homocysteine hydrolase; the resulting mixture After incubation, contact with adenosine deaminase, nucleoside phosphorylase, xanthine oxidase and a peroxidase, and determine the UV absorbance of the mixture. 23. The method of claim 1, wherein the sample is contacted with adenosine and an S-adenosyl homocysteine hydrolase; incubating the resulting mixture and contacting with a fluorophore-labeled S-adenosyl homocysteine or a fluorophore-labeled furura-6-thioether; contacting the resulting mixture with a monoclonal anti-S-adenosyl homocysteine antibody; and determining by fluorescence polarization the antibody-bound or non-antibody-labeled fluorophore, from which the original homocysteine content of the sample is deduced. 24. The method of claim 1, wherein the sample is contacted with adenosine and an S-adenosyl homocysteine hydrolase; incubating the resulting mixture and contacting with labeled S-adenosyl homocysteine or labeled furanose-6-thioether; contacting the resulting mixture with a monoclonal anti-S-adenosyl homocysteine antibody bound to a carrier matrix; washing the antibody-containing matrix; and the labeled compound bound to the antibody is photometrically determined from which the original homocysteine content of the sample is deduced. 25. The method of claim 24, wherein the labeled compound bound to the antibody is reacted to produce a chromophore which is then determined photometrically. 26. The method of claim 3, wherein the assay is performed by determining the precipitation or agglutination of the antibody / polyhaptene conjugates. An analytical kit for the determination of homocysteine in a sample according to the method of claim 1, comprising the kit - a homocysteine converting enzyme, - a substrate other than homocysteine for the enzyme-catalyzed reaction of homocysteine, a symbolizing agent, and - where appropriate, means for measuring the signal. 28. The kit of claim 27, wherein the homocysteine converting enzyme is S-adenosyl homocysteine hydrolase; S-adenosyl homocysteine labeled as a substrate for the enzyme; an anti-S-adenosyl homocysteine antibody optionally bound to a carrier matrix; and optionally, as a means for measuring the signal, a means for photometric determination of the homocysteine content of the sample or the detectable S-adenosyl homocysteine or a detectable derivative thereof. 29. The kit of claim 27, wherein the homocysteine converting enzyme is S-adenosyl homocysteine hydrolase; adenosine as substrate for the enzyme; an adenosine converting enzyme other than S-adenosyl homocysteine hydrolase; optionally an adenosine substrate for the adenosine converting enzyme; and as a means for measuring the signal, a means for generating a photometrically detectable response from a product of the adenosine converting enzyme or a co-substrate of adenosine. 30. The kit of claim 29, wherein the adenosine converting enzyme is adenosine kinase and the ATP, luciferin, and a luciferase are the response generating means. 31. The kit of claim 29, wherein the adenosine converting enzyme is adenosine deaminase and the means for generating a response is nucleoside phosphorylase, xanthine oxidase and a peroxidase. 32. The kit of claim 27, wherein the homocysteine converting enzyme is S-adenosyl homocysteine hydrolase; adenosine as substrate for the enzyme; an anti-S-adenosyl homocysteine antibody optionally bound to a matrix particle; a polyhapten for the antibody; and optionally a means for determining the signal by photometric determination of the agglutination or precipitation of the antibody / polyhaptene complexes. 33. The process of claim 2, wherein said hapten is a compound of formula (I) R 1 and R _{2 are the} same or different and are hydrogen or -OR 4, or R] and _R2 together represent an oxygen atom, R ₃ is amino or carboxyl and R 4 is a C 1-6 aliphatic group or a salt or ester thereof. 34. The method of claim 4, wherein said hapten is a labeled furanose-6-thioether wherein said labeled residue is a chromophore or fluorophore or radioactive atom and said thioether residue comprises a trimethylene thio residue. 35. The process of claim 34, wherein the labeled thioether is a labeled compound of formula (I) as defined in claim 33. 36. The method of claim 9, wherein said enzyme is an inactivated SAH hydrolase which is activated by contacting with a reducing agent. 37. The kit of claim 27, comprising an inactive SAH hydrolase in a first compartment and a reducing agent in a second compartment to produce an activated SAH hydrolase by mixing the contents of the two compartments. 38. The kit of claim 37, wherein the second compartment comprises dithiothreitol in an acidic medium.